Proteomic Analysis of Exosomes from Human Neural Stem Cells by Flow Field-Flow Fractionation and Nanoflow Liquid Chromatography−Tandem Mass Spectrometry

Abstract

Exosomes, small membrane vesicles secreted by a multitude of cell types, are involved in a wide range of physiological roles such as intercellular communication, membrane exchange between cells, and degradation as an alternative to lysosomes. Because of the small size of exosomes (30-100 nm) and the limitations of common separation procedures including ultracentrifugation and flow cytometry, size-based fractionation of exosomes has been challenging. In this study, we used flow field-flow fractionation (FlFFF) to fractionate exosomes according to differences in hydrodynamic diameter. The exosome fractions collected from FlFFF runs were examined by transmission electron microscopy (TEM) to morphologically confirm their identification as exosomes. Exosomal lysates of each fraction were digested and analyzed using nanoflow LC-ESI-MS-MS for protein identification. FIFFF, coupled with mass spectrometry, allows nanoscale size-based fractionation of exosomes and is more applicable to primary cells and stem cells since it requires much less starting material than conventional gel-based separation, in-gel digestion and the MS-MS method.

Classification, Functions, and Clinical Relevance of Extracellular Vesicles

“…Although FFFF is successfully applied to isolate exosomes from human neural stem cells (Kang et al, 2008), FFFF is not widely applied because it requires extensive optimization of the settings and is relatively expensive.…”

Exosomes as intercellular signalosomes and pharmacological effectors

“…Exosomes contain peripheral membrane proteins such as MHC I and II [2] integrins, transferring receptors [62], tetraspanins [63] which are active towards downstream signaling pathways in target cells, triggering for instance calcium signaling [64], MAPK activation [65], or NKG2D signalling [66][67][68]. Exosomes also contain active lipolytic activies such as phospholipases leading to the formation of bioactive lipid mediators (fatty acids and prostaglandins) which can interact with peripheral 11 G-protein coupled receptors and nuclear receptors in target cells [9].…”

Synaptic vesicles studied by dynamic light scattering

“…AFFF has been used for characterization and size fractionation of vesicles [22][23][24][25][26], including applications to biological vesicles [27,28]. The dispersed particles travel along a channel with a parabolic velocity profile in the principal flow direction.…”